Two-Dimensional Chromatographic Isolation of High Purity Erinacine A from Hericium erinaceus.

Abstract

A simple and robust two-dimensional chromatographic fractionation protocol for the isolation of the neuroprotective compound erinacine A from Hericium erinaceus is proposed. This production platform yielded 19.4 mg of erinacine A from approximately 130 g of mushroom material, with a chromatographic purity of 97.4%. The procedure includes extraction, concentration, fractionation, purification, and characterisation of the bioactive compound. The crude H. erinaceus extract was fractionated in the first dimension by normal-phase flash chromatography, and the fraction containing erinacine A was further purified in the second dimension by semi-preparative reversed-phase chromatography. This strategy utilises the orthogonality of the two chromatographic modes to effectively eliminate difficult impurities, including structural isomers and analogues of erinacine A. Complementary analytical approaches such as high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography with ultraviolet and tandem mass spectrometric detection (HPLC-UV-MS/MS) were employed to unambiguously confirm erinacine A in the isolated fractions, while HPLC with a charged aerosol detector (CAD) was used to determine its purity. Given the limited commercial availability and the high price of erinacine A, the described method offers a straightforward and cost-effective alternative to obtain this valuable compound for further research and applications.